Surface enhanced Raman gene probe and methods thereof

ABSTRACT

The subject invention disclosed herein is a new gene probe biosensor and methods thereof based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays.

This invention was made with Government support under contractDE-AC05-84OR21400 awarded by the Office of Health and EnvironmentalResearch, Department of Energy to Lockheed Martin Energy Systems, Inc.,and the Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to DNA gene probes, biosensors and methodsfor gene identification, particularly non-radioactive gene probes,biosensors and methods for oligonucleotide identification and moreparticularly to non-radioactive gene probes, biosensors and methodsbased on surface enhanced Raman scattering (SERS) label detection.

BACKGROUND OF THE INVENTION

There is currently strong interest in the development of nonradioactiveDNA probes for use in a wide variety of applications, such as geneidentification, gene mapping, DNA sequencing, medical diagnostics, andbiotechnology. Among the various methods for gene identification,technologies using radioactive labels are currently the most widelyused. Radioactive label techniques suffer from several disadvantageshowever. The principal isotope used, Phosphorus-32, has a limitedshelflife because it has a 14-day half-life. Secondly, because there isone principal label for gene probes, DNA can only be probed for onesequence at a time. Due to material limitations, probing immobilized DNAwith different ³² P-labeled sequences can only be performed a few (3-4)times. Therefore, the researcher must have idea about the sequence priorto probing. In addition to these inconveniences, the potential safetyhazard associated with use of radioactive materials makes the technologyundesirable. Shipping, handling and waste disposal of radioactivematerials are strictly regulated by federal and state guidelines.

Recently, luminescence labels such as fluorescent or chemiluminescentlabels have been developed for gene detection. Although sensitivitiesachieved by luminescence techniques are adequate, alternative techniqueswith improved spectral selectivities must be developed to overcome theneed for radioactive labels and the poor spectral specificity ofluminescent labels.

Spectroscopy is an analytical technique concerned with the measurementof the interaction of radiant energy with matter and with theinterpretation of the interaction both at the fundamental level and forpractical analysis. Interpretation of the spectra produced by variousspectroscopic instrumentation has been used to provide fundamentalinformation on atomic and molecular energy levels, the distribution ofspecies within those levels, the nature of processes involving changefrom one level to another, molecular geometries, chemical bonding, andinteraction of molecules in solution. Comparisons of spectra haveprovided a basis for the determination of qualitative chemicalcomposition and chemical structure, and for quantitative chemicalanalysis.

Vibrational spectroscopy is a useful technique for characterizingmolecules and for determining their chemical structure. The vibrationalspectrum of a molecule, based on the molecular structure of thatmolecule, is a series of sharp lines which constitutes a uniquefingerprint of that specific molecular structure. If the vibrationalspectrum is to be measured by an optical absorption process, opticalfibers must be used so that optical energy from a source is delivered toa sample via one fiber, and after passage through the sample, an opticalsignal generated by the exciting optical energy is collected by the sameor, more preferably, another fiber. This collected light is directed toa monochrometer/or a photodetector for analyzing its wavelength and /orintensity.

One particular spectroscopic technique, known as Raman spectroscopy,utilizes the Raman effect, which is a phenomenon observed in thescattering of light as it passes through a material medium, whereby thelight suffers a change in frequency and a random alteration in phase.When exciting optical energy of a single wavelength interacts with amolecule, the optical energy scattered by the molecule contains smallamounts of optical energy having wavelengths different from that of theincident exciting optical energy. The wavelengths present in thescattered optical energy are characteristic of the structure of themolecule, and the intensity of this optical energy is dependent on theconcentration of these molecules.

Raman spectroscopy is a spectrochemical technique that is complementaryto fluorescence, and has been an important analytical tool due to itsexcellent specificity for chemical group identification. Ramanspectroscopy provides a means for obtaining similar molecularvibrational spectra over optical fibers using visible or near infraredlight that is transmitted by the optical fibers without significantabsorption losses. In Raman spectroscopy, monochromatic light isdirected to a sample and the spectrum of the light scattered from thesample is determined. One of the major limitations of Raman spectroscopyis its low sensitivity. Recently, the Raman technique has beenrejuvenated following the discovery of enormous Raman enhancement of upto 10⁶ for molecules adsorbed on microstructures of metal surfaces.

Raman spectroscopy is a useful tool for chemical analysis due to itsexcellent capability of chemical group identification. One limitation ofconventional Raman spectroscopy is its low sensitivity, often requiringthe use of powerful and costly laser sources for excitation. However, arenewed interest has recently developed among Raman spectroscopists as aresult of observation that Raman scattering efficiency can be enhancedby factors of up to 10⁸ when a compound is adsorbed on or near specialmetal surfaces. Spectacular enhancement factors due to themicrostructured metal surface scattering process is responsible forincreasing the intrinsically weak normal Raman scattering (NRS). Thetechnique associated with this phenomenon is known as surface-enhancedRaman scattering (SERS) spectroscopy. The Raman enhancement process isbelieved to result from a combination of several electromagnetic andchemical effects between the molecule and the metal surface.

Deoxyribonucleic acid (DNA) is the main carrier of genetic informationin most living organisms. DNA is essentially a complex molecule built upof deoxyribonucleotide repeating units. Each unit comprises a sugar,phosphate, and a purine or pyrimidine base. The deoxyribonucleotideunits are linked together by the phosphate groups, joining the 3'position of one sugar to the 5' position of the next. The alternatesugar and phosphate residues form the backbone of the molecule, and thepurine and pyrimidine bases are attached to the backbone via the 1'position of the deoxyribose. This sugar-phosphate backbone is the samein all DNA molecules. What gives each DNA its individuality is thesequence of the purine and pyrimidine bases.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a nonradioactive gene probebiosensor based upon surface enhanced Raman scattering (SERS) labeldetection for identifying target oligonucleotide strands such asDeoxyribonucleic acid (DNA), Ribonucleic acid (RNA) and Peptide nucleicacid (PNA) in a variety of samples such as environmental samples orbiological samples. It is another object of the invention to provide aSERS gene probe biosensor for the identification of bacterial and viralgene sequences.

It is a further object of the invention to provide a SERS gene probedetection system for the detection and identification of biotargets suchas DNA, RNA and PNA in bacteria and viruses. It is yet another object ofthe invention to provide methods for using a SERS gene probe biosensorfor hybridization, detection and identification of hybridized targetoligonucleotides such as DNA, RNA and PNA in bacteria and viruses in avariety of samples such as environmental or biological samples. Furtherand other objects of the present invention will become apparent from thedescription contained herein.

SUMMARY

The subject invention is a new type of gene probe biosensor based onsurface enhanced Raman scattering label detection. The surface enhancedRaman (SER) gene probes do not require the use of radioactive labels andhave great potential to provide both sensitivity and selectivity. TheSER gene probe is used to detect DNA biotargets such as gene sequences,bacteria and viral oligonucleotide strands via hybridization tooligonucleotide strands complementary to the SER gene probe.

In accordance with one object of the invention, a SER gene probebiosensor comprises a support means, a SER gene probe having at leastone oligonucleotide strand labeled with at least one SERS label, and aSERS active substrate disposed on the support means and having at leastone of the SERS gene probe adsorbed thereon.

In accordance with another object of the invention, a SER gene probedetection system comprises a SERS active substrate having at least oneSER gene probe adsorbed thereon wherein the SER gene probe has at leastone oligonucleotide strand labeled with at least one SERS label. Thesystem further comprises an energy source, a means for transmittingoptical energy from an optical energy source to the SERS activesubstrate in order to generate a Raman signal, a means for collectingthe Raman signal and transmitting the Raman signal for detection, and ananalyzing means for detecting and processing the Raman signal.

In accordance with yet another object of the invention, a method forusing a SER gene probe for hybridization and detection to identifyhybridized target oligonucleotide strands comprising the steps of: a)preparing a sampling medium with immobilized oligonucleotide strands ofknown sequence adsorbed thereon wherein the immobilized oligonucleotidestrands are complementary to the target oligonucleotide strands; b)synthesizing SER gene probes wherein a SER gene probe comprises at leastone oligonucleotide strand of unknown sequence having at least one SERSactive label; c) preparing a SER gene probe solution comprising at leastone SER gene probe wherein the SERS label is unique to theoligonucleotide strand of a particular sequence; d) incubating thesample medium in an amount of SER gene probe solution sufficient enoughto hybridize the immobilized oligonucleotide strands on the samplemedium with the target oligonucleotide strands that are complementary tothe immobilized oligonucleotide strands, incubating for a time periodsufficient enough as for the SER gene probes to contact the immobilizedoligonucleotide strands and sufficient enough as for hybridization tooccur, thereby producing hybridized oligonucleotide material; e)removing the oligonucleotide strands that did not hybridize to theimmobilized oligonucleotide strands; f) recovering the hybridizedoligonucleotide material from the sampling medium; g) transferring to aSERS active substrate a small amount of the recovered hybridizedoligonucleotide material in an amount sufficient enough as to provide adetectable quantity of hybridized oligonucleotide material; and h)analyzing the SERS active substrate containing the hybridizedoligonucleotide material.

In accordance with still another object of the invention, a method forusing a SER gene probe for hybridization and direct detection toidentify hybridized target oligonucleotide strands comprising the stepsof: a) preparing a SERS active substrate having adsorbed thereon atleast one SER gene probe complementary to the target oligonucleotidestrand wherein the SER gene probe comprises at least one oligonucleotidestrand of known sequence labeled with a SERS label unique for the targetoligonucleotide strands of a particular sequence; b) introducing theSERS active substrate into a sample suspected of containing targetoligonucleotide strands and contacting the SER gene probe with thetarget oligonucleotide strands for a time sufficient enough as for thecontact to occur and hybridization to occur between the targetoligonucleotide strand and the complementary SER gene probe, therebyproducing hybridized oligonucleotide material; c) removing from the SERSactive substrate, remaining sample containing nonhybridizedoligonucleotide strands; and d) analyzing the SERS active substratecontaining the hybridized oligonucleotide material.

In accordance with still yet another object of the invention, a methodfor using a SER gene probe for detection and identification of targetDNA strands that have been amplified through Polymerase Chain Reactioncomprising the steps of: a) preparing a SERS active substrate havingadsorbed thereon two unlabeled DNA strands of known sequence, beingcomplementary to a target region of a target DNA strand, said target DNAstrand comprising double strands of DNA complementary to one another,and said SERS active substrate being disposed on a support means;

b) synthesizing two SER gene probes as primers wherein each of said SERgene probes comprises an oligonucleotide strand complementary to siteson the opposite DNA strands of said target DNA strand wherein eachprimer has a sequence which is identical to the 5' end of one DNA strandof said target DNA strand, each of said SER gene probes furthercomprises a SERS label attached to said oligonucleotide strand; c)heating said target DNA strand to a temperature sufficient fordenaturization of said double strands of said target DNA to occur toform single-stranded DNA templates; d) annealing said two primers tosaid DNA templates at a temperature ranging from 40°-60° C. wherein eachprimer binds to said complementary sequence at the 3' end of saidopposite DNA strand of said target DNA strand; e) adding DNA polymeraseto extend the DNA molecule through said target region of said target DNAstrand yielding amplified products, said amplified products being SERSlabeled amplified DNA segments; f) immersing said SERS active substratein a sample containing said amplified products; g) incubating said SERSactive substrate in said sample for a time sufficient enough as forhybridization between said SERS labeled amplified DNA segments and saidunlabeled DNA strands on said substrate to occur to completion and aSERS signal is detected; and h) analyzing said SERS signal.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the present invention, together with otherand further objects, advantages and capabilities thereof, reference ismade to the following disclosure and appended claims when read inconnection with the appended drawings, wherein:

FIG. 1 illustrates a SER Gene Probe Detection System.

FIG. 2 shows a diagram of the method of using a SER Gene Probe forhybridization and detection with the SER Gene Probe attached to thenucleotide strands to be identified.

FIG. 3a is a SERS Spectrum of only cresyl fast violet label.

FIG. 3b is a SERS Spectrum of cresyl fast violet label attached to 18deoxyribonucleotide oligomers, p(dT)₁₈.

FIG. 4a is a spectrum showing detection of the SER Gene Probe that hashybridized to a DNA fragment complementary to the probe.

FIG. 4b is a spectrum showing no SERS detection.

FIG. 5a shows the absorption spectrum of cresyl fast violet.

FIG. 5b shows the fluorescence spectrum of cresyl fast violet.

FIG. 6a is a diagram illustrating the method of direct detection withthe SER Gene Probe being attached to the SERS Active Substrate.

FIG. 6b illustrates the same direct detection method as FIG. 6a, exceptno hybridization occurs due to absence of target oligonucleotides.

FIG. 7 is a schematic diagram of a SER Gene Probe Fiberoptic Biosensor.

FIG. 8 shows a close-up view of the dotted area of the SER Gene ProbeFiberoptic Biosensor in FIG. 7 with SER Gene Probes being attached tothe SERS active substrate on the fiberoptic probe tip.

FIG. 9 shows an alternate embodiment to the SER Gene Probe FiberopticBiosensor of FIG. 8.

FIG. 10 is a schematic diagram of an array of SER Gene Probe FiberopticBiosensors each having a SERS active substrate with immobilized SER GeneProbes used for conducting simultaneous multiple assays.

FIG. 11 illustrates SER Gene Probes attached to a SERS active substrateon the surface of a Waveguide Biosensor.

FIG. 12 is a diagram of SER Gene Probes attached on a SERS activesubstrate on the surface of waveguide microsensor arrays withcharge-coupled devices or photodiode arrays.

FIG. 13 illustrates a method for using the SER gene probe in conjunctionwith Polymerase Chain Reaction to detect target oligonucleotide strands.

DETAILED DESCRIPTION OF THE INVENTION

The possibility of using Raman and/or SERS for in situ monitoring hasbeen reported in the past few years as well as development of efficientSERS active substrates for trace organic analysis in environmental andbiological applications. The SERS technique has also been applied totrace detection of pesticides, dyes, food products, and metabolites ofchemical exposure. The subject invention herein discloses the use of theSERS technique as a tool for detecting specific nucleic acid sequences.An example of a hybridization experiment using the SER gene probeillustrates the usefulness of this technology. Hybridization of anucleic acid probe to DNA biotargets such as gene sequences, bacteriaand viral DNA permits a very high degree of accuracy for identifying DNAsequences complementary to that probe.

Applicant's SERS gene probe technology can rapidly detect microorganismsfrom multiple environmental samples. Examples include detection ofSalmonella bacteria, the causative agent for food poisoning, during foodprocessing; detection of Legionaella bacteria, the causative agent forpneumonia, from water samples; detection of Giardlia lamblia, causativeagent for diarrhea, from water samples; and detection of Hepatitis virusfrom shellfish. Applicant's SERS gene probe biosensor can also have aglobal impact on biosensor technology in cancer detection. Applicant'sbiosensor is able to detect both DNA and RNA viruses and retroviruses inparticular which can play a part in transforming healthy cells intocancer cells. Examples of this global impact include the detection ofDNA viruses such as Papovavirus and its many strains which play a partin causing benign warts and carcinoma of uterine cervix worldwide. Thedetection of Hepadnavirus (Hepatitis B), which plays a part in causingliver cancer mainly in southeast Asia and tropical Africa, is nowpossible. Also, the detection of Herpesvirus, which plays a role incausing lymphocyte cancer and nasopharyngeal carcinoma mainly in westAfrica, southern China and Greenland. Applicant's probe can also make aglobal impact on the detection of the HIV-1 virus, the causative agentfor Kaposi's carcinoma and AIDS, worldwide. Another example is thedetection of human T-cells and HTLV-1 virus which play a part in adultT-cell mainly in Japan (Kyushu) and detection of leukemia/lymphoma,mainly in the West Indes. Applicant's probe is a sensitive DNA biosensorthat can detect viral diseases at the early stage of the infection. Yetother viruses and bacteria that can be detected are included in thecausative agents that play a role in AIDS, Lyme Disease, Rocky MountainSpotted Fever, Tuberculosis, Toxoplasmosis and Cancer. These bacteriaand viruses can dwell in numerous different mediums which can beanalyzed by Applicant's SERS gene probe biosensor. These differentmediums include bodily fluids, blood, sputum, cat feces, raw meat andother tissues.

Applicant's invention is a Surface Enhanced Raman (SER) gene probebiosensor used for the detection and identification of hybridizedoligonucleotide strands labeled with a SERS label wherein identificationof a target oligonucleotide is based on the detection of the SERS label.The SER gene probe biosensor comprises a support means, a SER gene probeand a SERS active substrate disposed on the support means. The SERSactive substrate has at least one SERS gene probe adsorbed onto thesubstrate. The SER gene probe has at least one oligonucleotide strandlabeled with at least one SERS label. Oligonucleotides include DNA, RNAand PNA. The oligonucleotide of the SER gene probe either has the SERSlabel attached to the strand or if two oligonucleotide strands are used,the SERS label can be intercalated between the two oligonucleotidestrands, enveloped by the oligonucleotide strands holding the label inplace. If the SERS label is attached to the oligonucleotide strand, thelabel can be attached either at the end of the strand or at any sitebetween the strand ends. More than one SERS label can be used to labelas long as it does not interfere with hybridization. Many different SERSlabels can be used. Examples of the different SERS labels that can beused include cresyl fast violet, cresyl blue violet, para-aminobenzoicacid, erythrosin and aminoacridine. Other SERS labels that can be usedthat are inert to hybridization are chemical elements or structures thatexhibit a characteristic Raman or SERS emission. These chemical elementsor structures include cyanide, a methyl group, a thiol group, achlorine, bromine, phosphorus and sulfur.

In one embodiment, Applicant's invention requires the oligonucleotidestrand to be labeled with a SERS label for detection and identification.In another embodiment, a SERS label can be entrapped or intercalatedinto a double strand of oligonucleotide. The label is a specificchemical group that can be detected using the SERS spectrographictechnique. Raman spectroscopy is a spectrochemical technique that iscomplementary to fluorescence, and is an important analytical tool dueto its excellent specificity for chemical group identification.Recently, however, there has been enormous Raman enhancement of up to10⁸ for molecules adsorbed on SERS active substrates, microstructures ofmetal surfaces. See, for example, D. J. Jeanmaire and R. P. Van Duyne J.Electronal. Chem., 84, (1977).

The SERS active substrate includes a support base having a roughenedmetal surface having a degree of roughness sufficient to induce the SERSeffect described above. The roughened surface is preferably formed byapplying a microparticle or microstructure layer to the surface of thesupport base and then depositing a metal layer onto the microstructurelayer. The roughened surface may be formed using conventionaltechniques, such as described in U.S. Pat. No. 4,674,878, incorporatedherein by reference.

For the SERS active substrate to be effective for detecting andidentifying a target oligonucleotide strand, the target oligonucleotidestrand must be in the vicinity of the roughened surface. An overcoat ofsilica, metal oxides, self-assembled organic monolayer layer or organicpolymer can be applied to the metallic microstructure layer. The coatingis applied to the roughened surface to sorb the oligonucleotide materialwhich are not easily adsorbed by the roughened surface and which arecapable of either penetrating into the coating or being attached ontothe coating. The oligonucleotide material thereby is adsorbed andbecomes positioned in the vicinity of the roughened surface and exhibitthe SERS effect. Thus, in essence, the coating serves to "alter" theadsorptivity of the roughened surface. The oligonucleotide materialimmobilized on the roughened surface of the SERS active substratecomprises either the labeled SER gene probe immobilized on the SERSactive substrate which later hybridizes with the target oligonucleotidestrand before analysis or it comprises an oligonucleotide strand ofknown sequence immobilized on the SERS active substrate which laterhybridizes to the SERS labeled target oligonucleotide strand of unknownsequence. Therefore, the SER gene probe can be immobilized on the SERSactive substrate before hybridization with a target oligonucleofidestrand FIG. 6 or the SER gene probe is attached to an knownoligonucleotide sequence complementary to a target oligonucleotidestrand and is hybridized to the immobilized oligonucleotide strand onthe substrate.

The coating may be an organic or inorganic sorbent material such assilica or self-assembled organic monolayer, or is an organic sorbentpolymer coating, such as polymethyl-methacrylate. Selection of thepolymer is based on the sorbtivity of the polymer for theoligonucleotide material to be identified. Selection criteria forcoatings may be based upon the desired physical (e.g., size selectivity,permeability), chemical (e.g. polarity, chemical selectivity),electrical, magnetic, nuclear radiation-hardening and biologicalproperties of the coating materials. Examples of other coating materialsinclude carnauba wax, ethyl cellulose, ethylene maleic anhydridecopolymer, methyl vinyl ether, octadecyl vinyl ether, phenoxy resin,poly 2-ethylhexyl methacrylate, poly (caprolactone), poly (caprolactone)triol, poly-1-butadiene, poly-n-butyl acrylate, poly-p-vinyl phenol,polybutadiene oxide, polybutadiene hydroxy terminated,polybutadiene-methylacrylated, polycutadiene acrylonitrile, polydecylacetate, polyethyl acrylate, polyethylene, polyethylene glycol methylether, polyhexyl methacrylate, poly 1 butene, polymethacrylate,polystyrene, polyvinyl butyryl, polyvinyl carbazone, polyvinyl chloride,polyvinyl isobutyl ether, polyvinyl methyl ether, polyvinyl stearate,and vinyl alcohol/vinyl/actate copolymer.

In most cases, oligonucleotides such as DNA have to be attached ontoSERS active substrates which can have as its support a glass microscopeslide, a surface of a fiberoptic probe biosensor, a fiberoptic probebiosensor array, a waveguide or waveguide microsensor arrays. Since mostof SERS coatings are based on silver or gold, the binding ofoligonucleotides on the metal surface can be based on thiol chemistry orother standard chemical binding methods. The thiols are known tostrongly chemisorb to silver and gold surfaces to form monolayers thatpossess supramolecular properties, as found in G. Whitesides and P.Laibnis, Langmuir, 6, 87-95, 1990, incorporated herein by reference.

If the overcoat is silica, the gene probe is bound to the silicacoating. The silica surface is derivatized with silane by incubation ina 2% 3-aminopropyl triethoxysilane (APTS) for 24 hr. at roomtemperature, washed in acetone and dried in vacuum. The silanyl groupsare activated by incubation in 1% glutaraldehyde in water for one hourat room temperature. Excess glutaraldehyde is removed by washing inwater and rinsing with phosphate buffered saline (PBS). Oligonucleotideprobe molecules containing amino linkers are attached to the silicasurface by incubating for 24 hr at 4° C. with a probe solution (e.g.,concentration 10 mg/ml). Unbound probe is washed away with PBS. A. Palet al, Analytical Chemistry, 67, 3154, 1995, is incorporated herein byreference.

The oligonucleotide strand of the SER gene probe is complementary to atarget oligonucleotide strand when the SER gene probe is immobilized onthe SERS active substrate. The SERS label is unique for a particulartarget oligonucleotide of a particular sequence that is characteristicof a particular bacteria or virus. So, if there are more than one SERgene probe utilized to assay for more than one particularoligonucleotide sequence characteristic of a particular bacteria orvirus, then each SER gene probe that is unique for a particular targetoligonucleotide strand will have a different, separate unique SERSlabel. Target oligonucleotide strands in a multiple assay having thesame sequence are designated for the same SERS label.

FIG. 1 is a schematic diagram of a SERS gene probe detection system 1.The system comprises an energy source 5, a bandpass filter 10, a mirror15, a SERS active substrate 20, the SERS active substrate 20 having SERgene probes 25, a collection of optics 30, a Raman holographic filter35, optical fiber 40, coupling optics 45, a detector 50 being a signalanalyzer and a data processor 55. The bandpass filter 10 and the mirror15 provide a means for transmitting optical energy from the energysource 5 to the SERS active substrate 20 to generate a Raman opticalsignal from the SER gene probe 25 being labeled with a SERS label. Thecollection of optics 30, the Raman holographic filter 35, the opticalfiber 40 and the coupling optics 45 provide a means for collecting theRaman optical signal and transmitting the signal for detection by asignal analyzer 50. The signal analyzer 50 and the data processor 55provide an analyzing means for detecting and identifying hybridizedtarget oligonucleotide strands.

Instrumentation for the experimental was as follows. Raman measurementswere conducted with a SPEX Model 1403 double-grating spectrometer (SPEXInc.) equipped with a thermoelectrically cooled gallium arsenidephotomultipler tube (RCA, Model C31034), operated in thesingle-photon-counting mode. Data storage and processing were handledusing a personal computer (PC) with SPEX Datamate software. Themonochromator bandpass was 2 cm⁻¹. Laser excitation was the 620-nm lineextracted from the emission band of a rhodamine 6G loaded dye laser(Coherent, CR-599-21) pumped by an argon ion laser (Coherent,Innova-70). Tuning of a birefringent filter plus the use of a bandpassfilter permitted a narrow excitation bandpass centered at 620 nm. Laserpower was 25 mW for all measurements. A right-angle geometry of thelaser excitation source and the scattered radiation was employed. SERSmeasurements were performed using two experimental systems. TheSPEX-based system was used to generate the basic SERS spectra. AnICCD-based system was used to generate spectra in hybridizedexperiments. In this system, the 632.8-nm line from a helium-neon laserwas used with an excitation power of approximately 5 mW. A bandpassfilter was used to spectrally isolate the 632.8-nm line before focusingonto the sample. Scattered radiation was collected with a two-lenssystem which efficiently coupled the collected radiation to a 600 μmdiameter silica fiber (NA-0.26, General Fiber Optics). Signal collectionwas performed at 180° with respect to the incident laser beam. A Ramanholographic filter was used to reject the Rayleigh scattered radiationprior to entering the collection fiber. The collection fiber was finallycoupled to a spectrograph (ISA, HR-320) which was equipped with ared-enhanced intensified charge-coupled device (RE-ICCD) detectionsystem (Princeton Instruments, RE-ICCD-576S). ICCD control and dataprocessing was enabled by a Princeton Instruments ST-130 control unitand CSMA software installed on a PC. Fluorescence measurements wereperformed with the Perkin-Elmer fluorimeter (Model LS-50).

The agglomerate-free alumina (0.1 μm nominal particle diameter) used toprepare the SERS substrates was provided by Baikowski Int. Corp. Thealumina was suspended in distilled Milli-Q Plus water by sonication.

The highest grade of reagents available were used. All solutions wereprepared with distilled, deionized Mifli-Q Plus water. All nucleic acidsolutions were sterilized by autoclaving or by filtration through a0.22-μm filter. Exposure of labeled DNA to light was minimized by usingopaque siliconized glassware, aluminum foil covering, or reducedroom-lighting conditions.

The following Example 1 describes the method depicted by FIG. 2.

EXAMPLE 1

SERS active substrates were prepared in the following manner. Arectangular glass strip (2.5 cm×1.25 cm; 1 mm thick) was cut from amicroscope slide that served as the support base. The glass strip wasthen cleaned with nitric acid, distilled water, and ethnology and driedusing a stream of dried air. Alumina microparticles were used to form amicrostructured surface. Three drops of a 5% aqueous suspension ofalumina (type 0.1 CR) were delivered and evenly spread on the glassstrip. The glass strip was then placed on a conventional spinning deviceto uniformly spread the alumina on the surface of the glass. The glassstrip was spun at 2000 rpm. Then, a 100-nm layer of silver was thermallyevaporated onto the alumina-coated glass strip in a vacuum evaporator ata pressure of 2×10⁻⁶ torr to form the metal layer with a deposition rateof 2 nm/s.

Preparation of the Nitrocellulose Blot

Oligonucleotides for binding to nitrocellulose were prepared fromhomopolymers of adenosine or thymidine. Samples were heated for 10minutes at 100° C., rapidly chilled on ice and diluted to 50% with 1MNaOH. Alkaline-treated DNA was then incubated for 30 minutes at roomtemperature before neutralization with the following solution: 0.5-MTris, 1M NaCl, 0.3M sodium citrate, 1M HCl. Samples were thenimmediately chilled on crushed ice. A nitrocellulose filter (Sigma) wascut into 3 mm×3 mm squares and turned onto virgin parafilm. DNA (25 μL)was loaded onto the nitrocellulose in 5 μL aliquots, added sequentiallyto the same spot, leaving sufficient time to absorb the material betweenadditions. The amount of DNA affixed to the membrane was 2.5 μg. Filterswere air dried for 2 h and then washed with 50 mL of SSC-20X solution(175.3 g of NaCl, 88.2 g of Na citrate in 1 L H₂ O; pH 7.0). Afterwashing the DNA loaded nitrocellulose, the filters were redried andbaked for 2 h at 75°-85° C. in a vacuum oven. FIG. 2 shows thenitrocellulose filter 2 with immobilized DNA 4 adsorbed thereon.

Synthesis of SER Gene Probes

Various DNA probes having different SERS labels were prepared. Solutionsof cresyl fast violet (Fluka), erythrosin, and aminoacridine (Sigma)were prepared at 0.15-0.25M concentration. Labeled oligonucleotides weresynthesized as 5'-phosphoramidates using a modification of the proceduredescribed by Chu et al Nucleic Acids Res., 1983, 11, 6513, incorporatedherein as a reference. Briefly, solutions of deoxyribonucleotideoligomers (either 9 or 18 residues in length; (Sigma) were converted to5'-phosphor-oimidazolide intermediates with 0.2M imidazole and 0.5M1-ethyl-3-(dimethylamino)propyl carbodiimide by incubation at 50° C. for3 h. The 5'-phosphorimidazolides were then reacted with equal volumes ofthe SERS active labels (e.g., cresyl fast violet dye) for 18 h at 50° C.Unreacted label was removed from the reaction mixture by gel mixture bygel filtration on Biospin 6 columns (Bio-Rad). The resultant labeledoligonucleofide samples 6 were concentrated by lyophilization.

Hybridization Procedures

Nitrocellulose filters 2 containing the DNA to be hybridized 4 wereplaced in siliconized 1.5 mL microfuge tubes. Distilled water was addedto the tube and was boiled in a water bath. The water was then removedby gentle aspiration. Negative controls consisted of labeled DNA thatwas not complimentary to the immobilized DNA.

The filters 2 were incubated overnight at 40° C. in hybridizationsolutions containing 2 ng/mL of the labeled probe 6. DNA which did nothybridize was removed by washing three times with SSPE-20X solution (174g of NaCl, 27.6 g of NaH₂ PO₄ in 1 L of H₂ O; pH 7.4) containing 0.1%SDS at room temperature.

Material which hybridized 4,6 to the nitrocellulose 2 was recovered asfollows. Filters were washed twice with 1 mL of SSC solution (0.1N NaOH)for 30 minutes at room temperature and three times with 1 mL of SSC,followed by vortexing. The wash buffer was aspirated, neutralized,pooled, and lyophilized before SERS analysis. The reconstitution volumewas 30 μL, and only 1 μL 4,6 was spotted onto the SERS substrate 8.

Referring to FIG. 2, an energy source 5 generates the exciting opticalenergy and the exciting optical energy is transmitted by a transmittingmeans 12 to the hybridized oligonucleotide material 4,6 on the SERSactive substrate 8. A Raman optical signal is generated and is collectedand transmitted by means 14 to a signal analyzer 16 for detection. Themeans for transmitting exciting optical energy includes filters andmirrors as well as optical fibers. The means for collecting andtransmitting the optical signal from the SERS active substrate include acollection of optics such as lenses, mirrors and/or filters as well asoptical fibers.

The method of FIG. 2 can also utilize microparticulates as the samplingmedium, wherein the oligonucleotide strands of known sequence areimmobilized onto the microparticulates. These microparticulates comprisemicrospheres, magnetic particles, magnetic particles coated with polymeror other microstructures. Then, instead of recovering the hybridizedoligonucleotide material from the sample medium and transferring a smallamount to a SERS active substrate, the amount transferred would includethe microparticulates with the hybridized material still attached. Theneed to recover the hybridized oligonucleotide material from the samplemedium is eliminated. When magnetic particles are used means forattracting the magnetic particulates can be used for the transferprocess.

FIG. 3a shows a SERS spectrum of a dye, cresyl fast violet (CFV), thatwas used in Example 1 for DNA labeling. The measurement was performedusing a silver-coated alumina substrate. The laser wavelength was 620nm, and the excitation power only 25 mW. The SERS spectrum of cresylviolet exhibits a series of narrow lines with the strongest at 590 cm⁻¹.This intense and sharp line can be attributed to the benzene ringdeformation mode. Another less intense but sharp line at 1195 cm⁻¹ couldbe related to benzene ring breathing vibrations. Another group of smallpeaks between 1000 and 1400 cm⁻¹ could be associated with aromatic ringsubstitution-sensitive modes. Finally several peaks, which couldcorrespond to benzene stretch vibrations, occur between 1500 and 1650cm⁻¹.

Cresyl fast violet, which was used as the model DNA label in Example 1,was covalently attached to a nucleic acid fragment consisting of 18deoxyribonucleotide oligomers of thymine, p(dT)₁₈. The SERS spectrum ofthis labeled DNA fragment is shown in FIG. 3b. The SERS-active substrateused for this figure is identical to that used to obtain the SERSspectrum of the CFV label alone in FIG. 3a. FIG. 3b demonstrates that itis possible to detect the spectral characteristic features of the CFVlabel even when it is bound to a large p(dT)₁₈ oligonucleotide gragment.Comparison of FIG. 3a and 3b indicates that the presence of theoligonucleotides induces a decrease in the SERS intensity of the CFVlabel, but the features of the label are still visible. Although thereis an increase of the background emission when the CFV label is attachedto the DNA fragment, the sharp peak associated with the label at 585cm⁻¹ remains the most prominent SERS line of the labeled DNA fragment. Aslight shift of this band is observed between the labeled dye (585 cm⁻¹,FIG. 3b) and the dye alone (590 cm⁻¹, FIG. 3a). Careful inspection ofFIG. 3b indicates that several other small peaks in the labeloligonucleotide system (445, 490, 675, 725, 1140, 1180 cm⁻¹, FIG. 3b)are similar to those detected in the label (450, 490, 675, 730, 1145,1195 cm⁻¹, FIG. 3a).

Different SERS labels can be used for different target oligonucleotidestrands of different sequences and different bacterial and viral types.SERS labels that can be used include cresyl fast violet, cresyl blueviolet, erythrosin, as well as aminoacridine. Other labels that exhibita characteristic Raman or SERS emission can also be used, as long as thelabel doesn't interfere with hybridization. The chemical structure orsubstituent to be used as a SERS label is not present in the originalnative DNA. Some chemical structures that can be used as a SERS labeland are inert to hybridization include cyanide (CN), thiol group (SH),chlorine (Cl), bromine (Br) and phosphorus (P). The SERS label can beattached at the end of the oligonucleotide strand or it can be disposedwith the oligonucleotide strand. More than one SERS label can be used ona given oligonucleotide strand. Another embodiment is one in which twooligonucleotide strands are used for the SER gene probe and the SERSlabel is disposed intercalated between the two strands. This particularembodiment provides the label to be held in place by the two strands.There is no attachment of the label on the oligonucleotide strands. Morethan one SERS label may be used for this embodiment as well.

To demonstrate the applicability of the SERS method in DNA gene probetechnology, a series of hybridization and SERS detection experimentswere performed. Hybridization, which involves the joining of a strand ofnucleic acid with its corresponding mirror image, is a powerfultechnique to identify DNA sequences of interest. FIG. 2 shows aschematic diagram of the hybridization and SERS detection of the probes.FIG. 4 shows the results obtained with the different samplesinvestigated. In these experiments, DNA fragments to be hybridized wereemployed, viz., p(dA)₁₈ oligonucleotides which are complementary to theSERS labeled p(dT)₁₈ probes discussed previously. The SERS labeledprobes that hybridized to the DNA oligomers attached on nitrocellulosewere recovered by washing the nitrocellulose and spotted on a SERSactive substrate (silver-coated alumina) for analysis. Negativecontrols, which consisted of labeled DNA that was not complementary topoly(dT)₁₈ fragments, for example, CFV-labeled p(dC)₉ oligonucleotides,were also analyzed. FIG. 4a shows clearly the SERS peak of the labeledp(dT)₁₈ probes that have hybridized to p(dA)₁₈. On the other hand,negative controls exhibit no SERS signals since the SER gene probes donot hybridize to the p(dC)₉ oligonucleotides (FIG. 4b).

Because SER gene probes rely on chemical identification, rather thanemission of radioactivity, they have a significant advantage overradioactive probes. SER gene probes are formed with stable chemicalswhich do not emit potentially dangerous ionizing radiation. Furthermore,the probes offer the excellent specificity inherent to Ramanspectroscopy. While isotope labels are few, many chemicals can be usedto label DNA for SERS detection. Potentially up to hundreds of differentSER gene probes can be constructed. A large number of probes withdifferent labels could be used to simultaneously probe one immobilizedDNA, PNA or RNA of interest.

Recently, luminescence labels (e.g., fluorescent or chemiluminescentlabels) have been developed for gene detection. Although sensitivitiesachieved by luminescence techniques are adequate, alternative techniqueswith improved spectral selectivities must be developed to overcome theneed for radioactive labels and the poor spectral specificity ofluminescent labels.

The spectral specificity of the SER gene probe is excellent incomparison to the other spectroscopic alternatives. For comparisonpurposes, the detection of the dye cresyl violet in UV absorption,fluorescence and SERS is compared. As shown in FIG. 5, the spectralbandwidth of cresyl fast-violet in UV absorption and fluorescence arebroad (typically 50-100 nm half-widths), whereas the bandwidth of theSERS spectrum of the same CFV dye is much narrower (<1 nm half-width,limited here by Raman spectrometer resolution; see FIG. 3). For thisreason, the Raman approach has a major advantage over the absorption orluminescence techniques. In a typical Raman spectrum, a spectralinterval of at least 2000 cm⁻¹ can provide 2000/2 or 10 availableindividual spectral "intervals" at any given time. Even allowing afactor of 10 due to possible spectral overlap, it should be possible tofind 10² labels that can be used for simultaneous detection of differentgene biotargets.

The method of FIG. 2 can incorporate a blotting procedure such as theSouthern blotting technique, the Western blotting technique or theNorthern blotting technique. A blotting technique provides an alternatemethod of recovering the hybridized oligonucleotide material from thenitrocellulose filter and transferring combined into one step, by simplyblotting the hybridized oligonucleotide material directly onto the SERSactive substrate. The small amount needed to be transferred onto thesubstrate is an amount sufficient enough as to provide a detectablequantity of hybridized oligonucleotide material to the SERS activesubstrate.

FIG. 6 illustrates a method for using SER gene probes as biosensors forhybridization and direct detection of the SERS label to identify thetarget oligonucleotide strands which are hybridized to the SER geneprobes. Referring to FIG. 6, a SERS active substrate 3 is prepared aspreviously described with the SER gene probe attached to the substrate.The SER gene probe 11 comprises at least one oligonucleotide strand 7 ofknown sequence labeled with a SERS label 9 which is unique for a targetoligonucleotide strand of a particular sequence. The oligonucleotidestrand of the SER gene probe is complementary to the targetoligonucleotide strand of which the SERS label is designated for.Following the hybridization step as previously described, the SER geneprobe 11 is hybridized with the target oligonucleotide 13 of which it isunique. FIG. 6a demonstrates hybridization 17 when targetoligonucleotides 13 are present in a sample. FIG. 6b demonstratesnon-hybridization due to the absence of target oligonucleotides. FIG. 6bshows that upon washing the oligonucleotides that are not complementaryto the SER gene probes 18 away, the SER gene probes 11 are leftunhybridized. Once hybridization has taken place and the substrateswashed to rid the unhybridized oligonucleotides 18, the SERS activesubstrate is analyzed 19. Due to the different hybridization state, theSERS gene probe in FIG. 6a (positive hybridization) and in FIG. 6b(non-hybridization), will exhibit different SERS signals.

FIG. 7 is a schematic diagram of a SER gene probe fiberoptic biosensor.Here, the SERS active substrate is disposed on the biosensor tip bybonding means such as adhesive or a SERS active substrate has beenformed on the biosensor tip making the tip the SERS active substrate.The formation of a SERS active substrate has been previously described.The SERS gene probe fiberoptic biosensor 73 is introduced into a sampleto be analyzed. Exciting optical energy from energy source 60 istransmitted by an optical fiber 70 through a bandpass filter 65 to theSER gene probe on the fiberoptic probe tip 73 generating a Raman opticalsignal from the SERS label. The optical signal is collected andtransmitted through the optical fiber 71 and coupling optics 75 to thespectrograph 80 to the detector 85 and to the controller 90. The energysource, bandpass filter, coupling optics, spectrograph, detector and thecontroller are a portable SERS monitor 100. FIG. 8 is a close-up view of73 in FIG. 7. FIG. 8a shows how exciting optical energy 63 enters theoptical fiber and is transmitted to the SER gene probe 11 on the SERSactive substrate 3, then how the optical signal 83 is collected and istransmitted back through the optical fiber and to a signal analyzer.FIG. 8b shows an alternate embodiment of FIG. 8a wherein the opticalfiber is tapered. FIG. 9 shows another embodiment of FIG. 8 where morethan one optical fiber is used, one for the excitation fiber and theother for the collection fiber to collect and transmit the signal to thesignal analyzer. Here, a mirror 95 is used to further direct the opticalenergy onto the SERS substrate 3 and onto the SER gene probe 11.

FIG. 10 shows an array of SER gene probe fiberoptic biosensors 73, 74,76, 77 each having a SERS active substrate with immobilized SER geneprobes attached to the biosensor tip. The device of FIG. 10 is capableof simultaneously performing multiple assays. Optical energy 63 from anoptical energy source is directed through a beam splitter 78 and onto afiberoptic array of multiple optical fibers 71 leading to separatefiberoptic biosensors 73, 74, 76, 77 having different SER gene probesunique for their different and separate complementary targetoligonucleotides. The optical signals generated from the SERS labels arecollected and transmitted by the array of optical fibers 71 to adetection array 98 where the signals are analyzed.

FIG. 11 shows a waveguide biosensor 84 that has a SERS active substrate3 on its surface. The SERS active substrate 3 has several SER geneprobes 11 attached thereto. Once again, the exciting optical energy froman energy source 86 is transmitted through the waveguide usingappropriate optics and filters to the surface of the waveguide to theSERS active substrate 3 and to the SER gene probes 11 to generate anoptical signal. The optical signal is transmitted using appropriateoptics and filters to the signal analyzer 88.

Since the SERS peaks are narrow, it is possible to detect several SERSlabels by spectral discriminating. Therefore, it is possible to attachseveral types of probes (probes for different SERS labels) on a singlewaveguide or a single optical fiber and detect several oligonucleotidestrands simultaneously.

FIG. 12 is a diagram of SER gene probes 11 attached on a SERS activesubstrate 3 on the surface of waveguide microsensor arrays 94 withcharge-coupled devices 99 or photodiode arrays. According to FIG. 12,optical energy from an energy source 86 is transmitted through thewaveguide 94 to the surface of the waveguide arrays and to the SERSactive substrates 3, to the SER gene probes 11 to generate an opticalsignal. The optical signal is transmitted to the two dimensionalcharge-coupled device detector (CCD) 99 and the data is sent for SERSrecording and processing 88. A CCD system is described by Yung-FongCheng et al, Appl. Spect., 44, 755-765, (1990), incorporated herein as areference. The system in the described in the reference, however, unitesCCD with capillary-zone electrophoresis rather than with a SERSbiosensor.

FIG. 13 illustrates a method for using the SER gene probe in conjunctionwith polymerase chain reaction (PCR) to detect target DNA strands. Inthis method, unlabeled DNA strands of known sequences 105 and 106,complementary to the double-stranded target DNA 112, respectively, areadsorbed onto a SERS active substrate 103 (step a, FIG. 13). The SERSactive substrate 103 is shown on the surface of support base 104. TheSER gene probes 115 and 120 are synthesized and used as SERS labeledprimers. One or two labeled primers can be used. In FIG. 13, theseprimers 115 and 120, of approximately 20 bases, are complementary tosites on the opposite DNA strands on either side of the double-strandedtarget DNA 111 and 113. Note that each primer hybridizes to the oppositestrand. Then, PCR is performed in the following manner: (step b) the DNAstrands are isolated and denatured to form single-stranded templates byheating to 90°-95° C. for approximately 1 minute. The two primers 115and 120 are annealed to the isolated single stranded DNA templates at40°-60° C. and then cooled for about 2 minutes and (step c) DNApolymerase (purified from the thermophilic bacterium Thermus aquaticus,Taq DNA polymerase) is added at about 72° C. for about 2 minutes,resulting in extension of the DNA molecule (amplification) through thetarget region 110 of the DNA strand 112. (Step d) Followingamplification by PCR, the SERS active substrate 103 on a support base104 is immersed and incubated in the sample containing the amplifiedproducts 125, which are the SERS labeled amplified DNA segments, for asufficient time as for hybridization between the SERS labeled amplifiedDNA segments and the unlabeled DNA strands 105, 106 on the substrate 103to occur to completion and a SERS signal can be detected. The SERSactive substrate having the unlabeled DNA strands complementary to thetarget DNA strand will detect the labeled amplified products. Followingmultiple cycles, there has been exponential amplification of the targetregion 110 containing SERS labeled primers 115 and 120. Therefore, theSERS labeled primers 115 and 120 have been amplified as well. Thisprocess can be repeated through any number of cycles to yield manycopies of the target sequence. While there has been shown and describedwhat are at present considered the preferred embodiments of theinvention, it will be obvious to those skilled in the art that variouschanges and modifications can be made therein, without departing fromthe scope of the invention defined by the appended claims.

What is claimed is:
 1. A Surface-Enhanced Raman (SER) gene probebiosensor comprising:a) a support means; b) a SER gene probe having twooligonucleotide strands labeled with at least one surface-enhanced RamanScattering (SERS) label intercalated between said two oligonucleotidestrands; and c) a SERS active substrate disposed on said support meansand having at least one of said SERS gene probe adsorbed thereon.
 2. TheSER gene probe biosensor of claim 1 wherein said SERS label comprisescresyl fast violet, cresyl blue violet, para-aminobenzoic acid,erythrosin or aminoacridine.
 3. A SER gene probe biosensor of claim 1wherein said SERS label comprises a chemical element or structure inertto hybridization, said chemical element or structure exhibiting acharacteristic Raman or SERS emission, said chemical element orstructure selected from the group consisting of CN, SH, CH₃, Cl, Br, Pand S.
 4. The SER gene probe biosensor of claim 1 wherein saidoligonucleotide strand comprises a strand of Deoxyribonucleic acid,Ribonucleic acid, or Peptide nucleic acid.
 5. The SER gene probebiosensor of claim 1 wherein said SERS active substrate furthercomprises an overcoat of silica, metal oxides, self-assembled organicmonolayer or organic polymer.
 6. The SER gene probe biosensor of claim 1wherein said oligonucleotide strand of said SER gene probe iscomplementary to a target oligonucleotide strand, said SER gene probehaving a SERS active label which is unique for said complementary targetoligonucleotide strand.
 7. The SER gene probe biosensor of claim 1wherein said support means comprises a fiberoptic probe having a probetip which supports said SERS active substrate, said fiberoptic probefurther having at least one optical fiber for transmitting excitingoptical energy from an energy source to said SER gene probe on said SERSactive substrate on said fiberoptic probe tip to generate a Ramanoptical signal and for collecting and transmitting said Raman opticalsignal to a signal analyzer.
 8. The SER gene probe biosensor of claim 1wherein said support means comprises an array of multiple opticalfibers, each of said optical fibers having a fiberoptic tip whichsupports a SERS active substrate labeled uniquely for a targetoligonucleotide complementary to said oligonucleotide strand of said SERgene probe, said array further having an energy source for generatingexciting optical energy and means for directing said exciting opticalenergy onto said optical fibers and said optical fibers transmittingsaid exciting optical energy to said SER gene probe on said SERS activesubstrate on said fiberoptic tip to generate a Raman optical signal,said array further having means for collecting and transmitting saidRaman optical signal to an array of signal analyzers.
 9. The SER geneprobe biosensor of claim 1 wherein said support means comprises awaveguide having a surface which supports said SERS active substrate,said waveguide further having means for transmitting exciting opticalenergy from an energy source to said surface and to said SER gene probeto generate a Raman optical signal and means for collecting andtransmitting said Raman optical signal to a signal analyzer.
 10. The SERgene probe biosensor of claim 1 wherein said support means comprises awaveguide having a surface which supports multiple SERS activesubstrates, each of said SERS active substrates having SER gene probeslabeled uniquely for a particular target oligonucleotide complementaryto said oligonucleotide strand of said SER gene probe, said waveguidefurther having means for transmitting exciting optical energy from anenergy source to said surface and to said SER gene probes to generate aRaman optical signal and means for collecting and transmitting saidRaman optical signal to a two-dimensional charge-coupled device foranalysis.
 11. A surface-enhanced Raman (SER) gene probe biosensordetection system comprising:a) a surface-enhanced Raman scattering(SERS) active substrate having at least one SER gene probe adsorbedthereon, said SER gene probe having two oligonucleotide strands labeledwith at least one SERS label, said SERS label intercalated between saidtwo oligonucleotide strands; b) an energy source; c) means fortransmitting optical energy from an optical energy source to said SERSactive substrate to generate a Raman signal; d) means for collectingsaid Raman signal and transmitting said Raman signal for detection; ande) an analyzing means for detecting and processing said Raman signal.12. A method for using surface-enhanced Raman a (SER) Gene ProbeBiosensor for direct hybridization, detection and identification ofhybridized target oligonucleotide strands comprising the steps of:a)preparing a surface-enhanced Raman scattering (SERS) active substratehaving adsorbed thereon at least one SER gene probe complementary tosaid target oligonucleotide strand wherein said SER gene probe comprisesat least one oligonucleotide strand of known sequence labeled with aSERS label unique for said target oligonucleotide strands of aparticular sequence; b) introducing said SERS active substrate into asample suspected of containing target oligonucleotide strands andcontacting said SER gene probe with said target oligonucleotide strandsfor a time sufficient enough as for said contact to occur andhybridization to occur between said target oligonucleotide strand andcomplementary said SER gene probe, thereby producing hybridizedoligonucleotide material; c) removing from said SERS active substrateremaining sample containing nonhybridized oligonucleotide strands; andd) analyzing said SERS active substrate containing said hybridizedoligonucleotide material.
 13. The method of claim 12 wherein saidoligonucleotide strand comprises Deoxyribonucleic acid, Ribonucleic acidor Peptide nucleic acid.
 14. The method of claim 12 wherein said SERgene probe comprises two oligonucleotide strands having at least oneSERS label intercalated between said oligonucleotide strands.
 15. Themethod of claim 12 wherein said SERS active substrate is disposed on afiberoptic probe having a probe tip which supports said SERS activesubstrate, said fiberoptic probe further having at least one opticalfiber for transmitting exciting optical energy from an energy source tosaid SER gene probe on said SERS active substrate on said fiberopticprobe tip to generate a Raman optical signal and for collecting andtransmitting said Raman optical signal to a signal analyzer.
 16. Themethod of claim 12 wherein multiple SERS active substrates are disposedon an array of optical fibers for performing multiple assays, each saidoptical fibers having a fiberoptic tip which supports a SERS activesubstrate labeled uniquely for a target oligonucleotide complementary tosaid oligonucleotide strand of said SER gene probe, said array furtherhaving an energy source for generating exciting optical energy and meansfor directing said exciting optical energy onto said optical fibers andsaid optical fibers transmitting said exciting optical energy to saidSER gene probe on said SERS active substrate on said fiberoptic tip togenerate a Raman optical signal, said array further having means forcollecting and transmitting said Raman optical signal to an array ofsignal analyzers.
 17. The method of claim 12 wherein said SERS activesubstrate is disposed on a waveguide having a surface which supportssaid SERS active substrate, said waveguide further having means fortransmitting exciting optical energy from an energy source to saidsurface and to said SER gene probe to generate a Raman optical signaland means for collecting and transmitting said Raman optical signal to asignal analyzer.
 18. The method of claim 12 wherein multiple SERS activesubstrates are disposed on a waveguide having a surface which supportssaid multiple SERS active substrates, each of said SERS activesubstrates having SER gene probes labeled uniquely for a particulartarget oligonucleotide complementary to said oligonucleotide strand ofsaid SER gene probe, said waveguide further having means fortransmitting exciting optical energy from an energy source to saidsurface and to said SER gene probe to generate a Raman optical signaland means for collecting and transmitting said Raman optical signal to atwo-dimensional charge-coupled device for analysis.